HLA Allelic and Genotypic Ambiguity Reduction in ImmPort

HLA Region Genomics Consortium 2010

HLA Allelic and Genotypic Ambiguity Reduction in ImmPort

Paula Guidry, Ph.D.BISC Team – UT Southwestern


HLA Typing Platforms and Ambiguity

HLA Typing Platforms SSOP – single-stranded oligonucleotide probe SSP – sequence-specific priming SBT – sequence-based typing SSCP – single-stranded conformation polymorphism

Allelic Ambiguity Typing methodology cannot distinguish between a set of

alleles Polymorphisms that distinguish these alleles are not

interrogated by the method Outside of exon 2 (class II) or exons 2 and 3 (class I) Sections of these exons not covered by the probes/primers

Genotypic Ambiguity Inability to determine phase in heterozygous samples


Ambiguity in HLA Typing Data


Ambiguity in HLA Typing Data

Allelic Ambiguity Reduction

Genotypic Ambiguity Reduction


Common and Well-Documented (CWD) HLA Alleles Allele has an estimated frequency of at least 0.001 in any

reference population Accurate frequency estimates require an allele to be observed

3+ times Studies with 1500 unrelated individuals (2n = 3000

chromosomes) can only accurately detect allele frequencies ≥ 0.001

OR Allele has been detected unambiguously in at least three

unrelated individuals but gene frequencies have not been estimated accurately

Rare HLA Alleles Alleles that are not CWD

Ambiguity Reduction Principles

CWD Alleles

Cano et al (2007). Common and Well-Documented HLA Alleles. Human Immunology 68: 392-417.


Meta-Analysis to Determine Worldwide HLA Allele Frequencies Data sources

13th IHW Anthropology/Human Genetic Diversity component 12th IHW Anthropology component Literature• 8 journals• Every issue from 1990 – 2007• 4 studies from other journals

AlleleFrequencies.net ≥ 20 individuals per population sample 497 population samples from around the world Total of 66,800 individuals


Regional CWD

Solberg et al (2008). Balancing Selection and Heterogeneity Across the Classical Human Leukocyte Antigen Loci: A Meta-Analytic Review of 497 Population Studies. Human Immunology 69: 443-464.


Reg-CWD HLA Alleles Allele is common and well-documented in a particular world

region*


Regional CWD

* Ignoring the past 1000 years of human migration

OTH – significantly admixed populations

Reg-Rare HLA Alleles Not reg-CWD Not detected in other

world regions

Neither Reg-CWD nor Reg-Rare Not reg-CWD Detected in other world

regions

HLA Allelic Ambiguity Reduction Heuristic

Steve Mack, Glenys Thomson,Rich Single






Phase 1

Eliminate others

Eliminate others

Are any of the alleles reg-CWD?

Are any of the alleles reg-CWD?

Elimination of less-probable alleles by CWD status

yes

no

Eliminate others

Eliminate others

Are any of the alleles

CWD?

Are any of the alleles

CWD?

yes

no

Eliminate others

Eliminate others

Are any of the alleles reg-Rare?

Are any of the alleles reg-Rare?

yes

Possible results: single allele call or multiple allele possibilities



Phase 2

Combine to G-code

Combine to G-code

Do any alleles belong to a common G-

code?

Do any alleles belong to a common G-

code?

yes

no

Combine to P-code

Combine to P-code

Do any alleles or G-codes belong to a common P-

code?

Do any alleles or G-codes belong to a common P-

code?

yes

Eliminate rare alleles

Eliminate rare alleles

Do the alleles contain any

G- or P-codes?

Do the alleles contain any

G- or P-codes?

yes

Possible results: single G- or P- call, multiple G- or P- calls or multiple alleles not related by G- or P- code

Reduction of multiple alleles to G- or P-codes



Phase 3

User determines most likely

allele

User determines most likely

allele

Are the alleles part of the same allele

family?

Are the alleles part of the same allele

family?

no

yes

Eliminate the lower frequency

alleles

Eliminate the lower frequency

alleles

Is the ratio of MRWAFs* for any pair of

these alleles > 9.49999?

Is the ratio of MRWAFs* for any pair of

these alleles > 9.49999?

yes

Keep the allele/code with the lowest 2nd field (third

and fourth digits)

Keep the allele/code with the lowest 2nd field (third

and fourth digits)

Reduction of alleles in the same family by frequency

no

Are there still multiple

alleles in the set?

Are there still multiple

alleles in the set?

yes

*MRWAF is the mean regional weighted allele frequency: mean frequency for an allele in a given region, weighted by sample sizes

(2n)


Outcome of HLA Allelic Ambiguity Reduction

HLA Genotypic Ambiguity Reduction

Heuristic

Steve Mack, Glenys Thomson,Rich Single






Phase 1

Reduction of sub-genotypes by CWD status

Eliminate others

Eliminate others

Are any sub-genotypes

reg-CWD/reg-CWD?


reg-CWD/reg-CWD?

yes

no

Eliminate others

Eliminate others


reg-CWD/CWD or

CWD/CWD?


reg-CWD/CWD or

CWD/CWD?

yes

no

Eliminate others

Eliminate others

Are any G/G, P/P, G/P,

G/reg-CWD, G/CWD, P/reg-CWD, P/CWD?

Are any G/G, P/P, G/P,

G/reg-CWD, G/CWD, P/reg-CWD, P/CWD?

yes

no



Phase 1, continued

Reduction of sub-genotypes by CWD status

Eliminate others

Eliminate others

Are any reg-CWD/reg-

Rare, CWD/reg-

Rare, G/reg-Rare or P/reg-

Rare?

Are any reg-CWD/reg-

Rare, CWD/reg-

Rare, G/reg-Rare or P/reg-

Rare?

yes

no

Eliminate others

Eliminate others


reg-Rare/reg-Rare?


reg-Rare/reg-Rare?

yes



Phase 2

Reduction of sub-genotypes by zygosity and allele numbers

Keep homozygous sub-genotypes

and sub-genotypes

containing these alleles

Keep homozygous sub-genotypes

and sub-genotypes

containing these alleles

Are any of the sub-

genotypes homozygou

s?

Are any of the sub-

genotypes homozygou

s?

yes

noKeep the sub-

genotype with the lowest sum of the 2nd field (third and

fourth digits)

Keep the sub-genotype with the lowest sum of the 2nd field (third and

fourth digits)


Outcome of HLA Allelic and Genotypic Ambiguity Reduction



ImmPortHLA Ambiguity Reduction Tool

www.ImmPort.org





Example HLA Typing Results File Ready for Ambiguity Reduction in ImmPort





Ambiguity Reduction Input File

Allelic Ambiguity Reduction Output Genotypic Ambiguity Reduction Output


Acknowledgements

Steven J. Mack , Ph.D.(CHORI)

Nishanth MarthandanThomas Smith, Ph.D.Patrick Dunn, Ph.D.John CampbellJeff Wiser

Richard Scheuermann, Ph.D.

David Karp, M.D., Ph.D.

Glenys Thomson, Ph.D. (Berkeley)

Richard Single, Ph.D. (UVM)

www.ImmPort.org



MRWAF Analysis

MRWAF is the mean regional weighted allele frequency:

mean frequency for an allele in a given region, weighted by sample sizes (2n)

If all alleles have MRWAF = 0 or all pairs have MRWAF ratio ≤ 9.49999, then these are passed to the next step

Documents

HLA Allelic and Genotypic Ambiguity Reduction in ImmPort